Abstract 4020: Wnt and EGFR crosstalk: Evidence for a bi-directional feedback loop important in maintaining the proliferative phenotype of normal urothelial cells

Abstract

Abstract Introduction The urothelial lining of the bladder is normally mitotically quiescent, but has a high capacity for self-renewal in response to injury. This regenerative ability serves to maintain urinary barrier function during normal homeostasis and offers a primary target for deregulation in cancer. Previous work in our laboratory has implicated autocrine signaling through EGFR activation but the role of other regulatory pathways remains poorly understood. There is some evidence that Wnt signaling may play a role in bladder cancer by promoting cell proliferation through β-catenin. In the absence of Wnt signaling, β-catenin is sequestered by the destruction complex and is targeted for degradation via the proteosome. When the Wnt signaling cascade is activated, the destruction complex is inactivated, allowing β-catenin to translocate to the nucleus, interact with specific transcription factors (TCF/LEF) and drive proliferation. The aim of this study was to assess whether Wnt signalling is functional in normal human urothelial (NHU) cells, and analyze the effects of Wnt and EGFR downstream signaling and potential crosstalk on proliferation. Materials and Methods NHU cells were grown as finite lifespan cell lines in proliferating monolayer cultures. Destruction complex antagonists were used to activate the Wnt pathway. Proliferation assays, immunofluorescence labeling, and TOPFLASH luciferase reporter assays were used to assess NHU responses. Pathway crosstalk was analyzed using shRNA targeted to β-catenin, as well as antagonists of the EGFR signaling pathways. Results Pharmacological inhibition of the destruction complex or activation of the Wnt pathway with exogenous Wnt3a ligand led to nuclear accumulation of β-catenin and an increase in TCF/LEF transcription factor activity. These effects were only apparent when autocrine EGFR signaling was blocked. Knockdown of β-catenin led to significant reduction in phospho-ERK and a modest increase in phospho-AKT. Conclusion Wnt signalling can be activated in NHU cells, verifying the presence of a functional pathway. Activation of the pathway is however masked by EGFR signaling, indicating pathway crosstalk. The effect of β-catenin knockdown on phospho-ERK indicated a bi-directional, positive feedback loop between the Wnt and EGFR signaling cascades in NHU cells. This work identifies the Wnt signaling pathway as an important signaling cascade in regulating the regenerative phenotype of NHU cells and indicates at least two pathways of regulation that may be circumvented in urothelial carcinogenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4020.